US6217875B1 - Inhibitors of lipoxygenase - Google Patents
Inhibitors of lipoxygenase Download PDFInfo
- Publication number
- US6217875B1 US6217875B1 US09/053,064 US5306498A US6217875B1 US 6217875 B1 US6217875 B1 US 6217875B1 US 5306498 A US5306498 A US 5306498A US 6217875 B1 US6217875 B1 US 6217875B1
- Authority
- US
- United States
- Prior art keywords
- perilla
- seed
- lipoxygenase
- composition
- luteolin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- SCZVLDHREVKTSH-UHFFFAOYSA-N COC1=CC(C2=CC(=O)C3=C(C=C(O)C=C3O)O2)=CC=C1O Chemical compound COC1=CC(C2=CC(=O)C3=C(C=C(O)C=C3O)O2)=CC=C1O SCZVLDHREVKTSH-UHFFFAOYSA-N 0.000 description 1
- IQPNAANSBPBGFQ-UHFFFAOYSA-N O=C1C=C(C2=CC=C(O)C(O)=C2)OC2=C1C(O)=CC(O)=C2 Chemical compound O=C1C=C(C2=CC=C(O)C(O)=C2)OC2=C1C(O)=CC(O)=C2 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/535—Perilla (beefsteak plant)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- This invention relates to inhibitors of lipoxygenase.
- This invention in regard to inhibitors of lipoxygenase, is intended for application in medicines regulating or preventing allergic diseases, inflammatory responses or the like. It is intended for use in connection with non-medical supplies, such as cosmetics or food additives and so forth.
- Arachidonic acid an essential fatty acid, exists inside the body as one compound of cell walls. Once arachidonic acid is stimulated, it is separated from phospholipid by the action of phospholipase. The separated arachidonic acid is metabolized by arachidonic acid metabolic enzymes e.g. lipoxygenase, then changed to substances such as prostaglandin (PG) which is associated with inflammatory responses or the like, *thromboxane (TX) which is associated with the formation of thrombus, and leukotriene (LT) and *lipoxyne, which induce allergic reactions. (See FIG. 1 ).
- PG prostaglandin
- TX *thromboxane
- LT leukotriene
- *lipoxyne which induce allergic reactions.
- arachidonic acid metabolic enzymes create substances which are the source of compounds that are associated with circulatory and allergic diseases, inflammation and so forth. Therefore, in order to regulate or prevent these diseases, it may be effective to inhibit the metabolism of arachidonic acid by enzyme action.
- 5-lipoxygenase metabolizes arachidonic acid into 5-hydroxy-6,8,10,14-eicosatetraenoic acid (5-HETE), and induces the formation of leukotriene (LT) which is associated with allergic diseases, inflammatory responses and asthma.
- 5-lipoxygenase is an enzyme having an arachidonic acid radical that metabolizes into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). This compound is associated with arteriosclerosis, allergic diseases and metastasis of cancer.
- Caffeic acid and quercetine or the like are well recognized as natural inhibitors of lipoxygenase. These substances, however, have not come into practical use as enzyme inhibitors. On the other hand, it is known that very few substances show significant inhibitory activity against 12-lipoxygenase. Other polyphenol group members are natural inhibitors of the activity of lipoxygenase. However, the inhibitory activity of these materials is not sufficient to produce satisfactory results in alleviating disease conditions. Further, it is difficult to cost-effectively purify enzyme inhibitors from natural substances.
- Perilla (frutescens) seed is a year-round plant belonging to the perilla (crispa) family. It is indigenous to East Asia and is classified as an oil crop. Perilla (crispa) typically has a square-shaped stem, oval-shaped opposed leaves and small white flowers that bloom in the summer season. Its seeds are a little larger than those of perilla (crispa) and are harvested in the autumn.
- perilla oil Oil obtained from perilla (frutescens) seed is known as perilla oil, and is used as an ingredient in food or paint. Also, oil cake is used as fertilizer or feed. It has already been reported that perilla leaves contain physiologically active substances, however, there have been few studies of perilla (frutescens) seed.
- Japanese Published Examined Patent Application No. 8-510735 describes the inhibitory activity of apigenin on 5-lipoxygenase.
- An imporantant object of this invention is to provide means to significantly inhibit the activities of 5-lipoxygenase and 12-lipoxygenase in relation to the metabolism of arachidonic acid.
- Another object of this invention is to extract active inhibitors of lipoxygenase from natural substances that may be used to regulate and prevent allergic diseases, inflammatory responses, circulatory diseases and metastasis of cancer.
- one aspect of this invention is the use of luteolin as an active compound to inhibit the enzyme action of lipoxygenase.
- Another feature of this invention is the use of chrysoeriol as an active compound to inhibit the enzyme action of lipoxygenase.
- Still another feature of this invention is the compounding of inhibitors of lipoxygenase from alcoholic extracts of perilla (frutescens) seed.
- These extracts comprise one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin.
- inhibitors of the enzyme action of lipoxygenase can be prepared from the alcoholic extract of perilla (frutescens) seed, including one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester, and apigenin.
- This alcoholic extract is suitably subjected to partition by ethyl acetate and water (two layers) and the fraction that partitions to the ethyl acetate layer is the desirable inhibitory fraction.
- defatted perilla frutescens
- perilla crispa
- the inventors have carried out various experiments on extracts of perilla (frutescens) seed, and have thereby discovered that an ethanol extract of perilla (frutescens) seed has a strong inhibitory effect against the enzyme action of 5-lipoxygenase and 12-lipoxygenase.
- the inventors have discovered that the luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and/or apigenin in an alcoholic extract of perilla (frutescens) seed strongly inhibit lipoxygenase enzyme activity. It was found that luteolin especially has an outstanding inhibitory activity against lipoxygenase. The outstanding inhibitory activity of luteolin on lipoxygenase, together with the lesser inhibitory activity of chrysoeriol, has only come to light through the research of these investigations.
- alcoholic extracts of other seeds also contain substances that effectively inhibit the enzyme activity of lipoxygenase, i.e. luteolin, chrysoeriol. rosemarinic acid and rosemarinic acid methyl ester.
- luteolin exists as a glycoside generally in the bean family i.e. digitalis, etc. Also, it is known that luteolin has physiological activity as an anti-oxidant, a hyaluronidase inhibitor, and so forth.
- luteolin when using luteolin as an active compound for its lipoxygenase inhibitory activity, it can be extracted or purified from the rind of citrus fruits as well as from perilla (frutescens) seed or perilla (crispa) seed and leaves.
- perilla frutescens
- perilla crispa
- chrysoeriol having physiological activity as an anti-cancer agent or antibiotic.
- it in order to use chrysoeriol as an active compound for its lipoxygenase inhibitory activity, it can be extracted or purified from perilla (frutescens) seed or perilla (crispa) seed and leaves.
- ethanol as solvent/extractant for the extraction of luteolin and chrysoeriol from perilla (frutescens) seed and perilla (crispa) seed.
- the use of ethanol allows the extracts to be available in formulations that are useful for both internal and external application.
- ethyl acetate, acetone, methanol, butanol and so forth can also be used as the extractant.
- an alcohol concentration between about 70 and 85% (v/v). If the concentration is less than about 70% (v/v), effective substances will not be sufficiently extracted, and if concentration is greater than about 85% (v/v), perilla (frutescens) seed oil easily dissolves into the alcohol. In order to improve the extraction of the active compound from seed, it is preferable to use repeated alcoholic extractions at various levels of concentration.
- Inhibitory substances obtained by alcoholic extraction of perilla (frutescens) seed and perilla (crispa) seed normally contain luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin. It is possible that, under different conditions of extraction, some or any of these substances may not be recovered. However, recovery of at least one of these substances is sufficient to inhibit the effects of lipooxygenase.
- alcoholic extracts of perilla (frutescens) seed or perilla (crispa) seed are best partitioned by ethyl acetate and water is that such a subsequent extraction produces a higher concentration of the active compound.
- a high concentration of luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester concentrates in the layer of ethyl acetate, while non-active compounds such as glycoside, etc., gather in the water layer. Therefore, the active compound can be more efficiently concentrated by using ethyl acetate to capture these effective compounds.
- compositions by directly compounding the aforementioned alcoholic extract and ethyl acetate partitioned compounds as inhibitors of the enzyme activity of lipoxygenase.
- alcoholic extraction using defatted perilla (frutescens) seed and perilla (crispa) seed provides a high concentration of active compounds. If the fat of the perilla (frutescens) seed and of the perilla (crispa) seed is extracted by treatment with organic solvents, the extracted oil contains only a small amount of physiologically active compounds, such as luteolin and chrysoeriol, because these active compounds are concentrated in the defatted fraction.
- Hexane for example, is appropriate as an organic solvent for defatting these seeds because the thus extracted oil can be used for food.
- the entire extract, oil and hexane or the like, from defatted perilla (frutescens) seed can also be used as an ingredient of foods and so forth.
- the substance extracted from defatted perilla (frutescens) seed is to be used for external application only, it would be possible to use not only hexane but also other non-polar solvent.
- inhibitors of the enzyme activity of lipoxygenase may be used in medical and non-medical applications. Exemplary non-medical applications include, among others: cosmetics, ingredient of foods and so forth. Since inhibitors of lipoxygenase, as described in this invention, exhibit effective inhibitory activity against the role of lipoxygenase in allergy and inflammation, it will also be appropriate to use them in anti-allergic or anti-inflammatory drugs or medicines.
- inhibitors of the enzyme activity of lipoxygenase may be administered either orally or by other means, such as topically.
- oral administration they can be given in tablet, granule, small grain, or powder form.
- inhibitors of lipoxygenase can be given by injection, intravenous drip, as solid, in suspension, in a viscoelastic fluid i.e. as a suppository that can be absorbed through the mucous membrane, by local topical application to internal or external organic tissue, or by other external administration i.e. intradermal, hypodermic, intramascular and intravenous injection, local topical application, spray, suppository, or injection to the bladder and so forth.
- the dose to be administered may fluctuate in accordance with administrative method, seriousness of the condition, age of the patient, condition and general situation of disease, stage of disease and so on. Normally, adults can take about 0.5 to 5,000 mg of active compound a day and children preferably can take about 0.5 to 3,000 mg of active compound a day.
- the concentration of inhibitors of lipoxygenase can be varied according to the type of administration. In case of oral administration or absorption through the mucous membrane, the concentration can be approximately 0.3 to 15.0wt %, and in case of non-oral administration, it can be approximately 0.01 to 10 wt %. It should be understood that the above mentioned amount is only exemplary and it is within the scope of this invention to vary the concentration and dosage in accordance with prevailing conditions.
- inhibitors of lipoxygenase of this invention When the inhibitors of lipoxygenase of this invention are to be used for medical or non-medical supplies or cosmetics, they may be used together with various common components of these formulations.
- These conventional ingredients are exemplified by:
- Oils animal oil, vegetable oil, mineral oil, ester oil, wax oil, silicon oil, higher alcohols, phospholipid, fatty acids, etc.
- Vitamins vitamin A, vitamin B, folic acid, nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D, vitamin E, ferulic acid, ⁇ -orizanol etc.
- Ultraviolet absorbents p-aminobenzoic acid, anthranil, salicylic acid, coumrin, benzotriazol, tetrazol, imidazoline, purimidine, dioxane, furan, pyrone, camphor, nucleic acid, allantoin and their conductors, amino acid compound, siconin, baicalin, baicalein, berberine, etc.
- Anti-oxidants ester stearate, nordihydroguaseleten acid, dibutyl hydroxytoluene, butyl hydroxyanisole, para hydroxyanisole, gallic acid propyl ester, sesamol, sesamolin, gossypol, etc.
- Thickeners hydxyethyl cellulose, ethyl cellulose, carboxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium, hydroxypropyl cellulose, nitrocellulose, polyvinyl alcohol (PVA), polyvinyl alcohol methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylic acid, carboxyvinyl polymer, gum arabic, tragacanth gum, agar-agar, casein, gelatin, pectin, starch, alginic acid and its salts.
- PVA polyvinyl alcohol
- PVA polyvinyl alcohol methyl ether
- polyvinyl pyrrolidone polyvinyl methacrylate
- polyacrylic acid carboxyvinyl polymer
- gum arabic gum arabic
- tragacanth gum agar-agar, casein, gelatin, pectin, starch, al
- Moisture retention agents propylene glycol, 1,3 butylene glycol, polyethylene glycol, glycerol, chondroitin sulfate, hyaluronic acid, sodium lactate, etc.
- the above mentioned materials can be used in medical or non-medical supplies such as cream, ointment, face lotion, body lotion, milky lotion, pack, oil, soap, including medicated soap, cleansing soap, bath aromatics, shampoo, hair conditioner, sprays. Also, they can be used in sanitary products, non-woven fabrics that are useful for tissues, wet wipes and the like, composites for oral use in case of stomatitis and so forth.
- lipoxygenase inhibitors as described in this invention are to be used in food additives, they can be also used in general foodstuffs such as confectionery, noodles, soup, beverages, and health foods including nutritional supplements.
- inhibitors of lipoxygenase enzyme activity can be used together with powdered cellulose, and can be spray dried or freeze-dried. Such a powder, granule, tablets or solution can easily be mixed with foods.
- 5-HETE which is a metabolite of 5-lipoxygenase, produces leukotriene and thereby induces allergic reactions or inflammatory responses.
- 12-HETE which is metabolite of arachidonic acid by the action of 12-lipoxygenase, is known to be a physiologically active substance in regard to arteriosclerosis or metastasis of cancer, Therefore, it is possible to regulate or prevent allergic diseases, inflammatory responses, circulatory diseases, metastasis of cancer, or the like by inhibiting these substances selectively.
- inhibitors of lipoxygenase can be obtained from defatted perilla after perilla (frutescens) seed oil for food has been collected. This means that defatted perilla (frutescens) seed can provide another resource.
- This invention provides active inhibitors of the enzyme activity of lipoxygenase. These inhibitors are obtained from natural substances and have significant inhibitory activities against both 5-lipoxygenase and 12-lipoxygenase. These inhibitors are available to regulate or prevent allergic reactions, inflammatory responses, circulatory problems and metastasis of cancer.
- a feature of the lipoxygenase inhibitors as described in this invention is that at least one of luteolin or chrysoeriol are used as the active compound.
- lipoxygenase inhibitors of this invention is the recovery thereof from alcoholic extract of perilla (frutescens) seed containing one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin.
- the aforementioned alcoholic extract is partitioned with ethyl acetate and water, and especially effective inhibitors are obtained from the ethyl acetate layer.
- Defatted perilla (frutescens) seed may preferably be substituted for whole perilla (frutescens) seed.
- Perilla (crispa) seed can be substituted for or used with the aforementioned perilla (frutescens) seed.
- FIG. 1 shows the physiological action of arachidonic acid metabolic enzyme.
- FIG. 2 is a flow chart to describe the method for isolation of inhibitors of lipoxygenase in accordance with the examples in this invention.
- FIG. 3 shows the correlation between the concentration of isolated luteolin and inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the embodiments in this invention.
- FIG. 4 shows the correlation between the concentration of isolated chrysoeriol and inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the embodiments of this invention.
- FIG. 5 shows the correlation between the concentration of ethanolic extraction and ethyl acetate fraction, and the inhibition of 5-lipoxygenase in accordance with the embodiments in this invention.
- FIG. 6 shows the correlation between the concentration of ethanolic extraction and ethyl acetate fraction, and the inhibition of 12-lipoxygenase in accordance with the embodiments in this invention.
- FIG. 7 shows the correlation between the concentration of isolated rosemarinic acid and the inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the comparisons in this invention.
- FIG. 8 shows the correlation between the concentration of isolated rosemarinic acid methyl ester and the inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the comparisons in this invention.
- crushed perilla (frutescens) seed was extracted (defatted) with hexane, and, after separation of the fat fraction, the residue of defatted perilla (frutescens) seed was then extracted by refluxing with 80% aqueous ethanol.
- the concentrated ethanolic extract was partitioned by hexane and 80% aqueous methanol.
- the 80% aqueous methanolic layer was evaporated to dryness and then partitioned between ethyl acetate and water. After separating the ethyl acetate layer from the water layer, some of the ethyl acetate solvent was distilled off, and a fraction of product in a smaller quantity of ethyl acetate was thus obtained.
- Embodiment 1 and 2 The pure luteolin and pure chrysoeriol isolated from perilla (frutescens) seed in this manner are respectively designated Embodiment 1 and 2.
- Embodiment 3 The ethanolic extraction (as illustrated in FIG. 2) obtained by evaporation of solvent was designated Embodiment 3, and the ethyl acetate layer obtained by partitioning the ethanolic extraction fraction was evaporated with the solvent, and then the fraction in the ethyl acetate was designated Embodiment 4.
- PMNL Peritoneal polymorphonuclear leukocytes
- Embodiments 1 to 4 Four to six testing solutions with different levels of concentrations were prepared for Embodiments 1 to 4. 0.02 mL Of enzyme solution (2 mg protein/mL) and [1l- 14 C] arachidonic acid (0.05 Ci) were incubated with each testing solution (0.02 mL) and Ca 2+ and ATP at 37° C. for 5 min.
- Embodiments 1 to 4 showed significant inhibitory activities against 5-lipoxygenase.
- Embodiment 1 luteolin
- Comparison 1 rosemarinic acid
- Comparison 2 rosemarinic acid methyl ester
- Embodiment 4 (ethyl acetate fraction) showed even more significant inhibitory activity than that of Embodiment 3 (ethanolic extraction). The reason for this phenomenon seems to be that active compounds are concentrated in the ethyl acetate layer by eliminating less active water and oil soluble substances from the ethanolic extract. Also, the inhibitory activities of Embodiment 3 (ethanolic extraction) and 4 (ethyl acetate fraction) seem to be correlated with their luteolin content.
- Table 1 shows the IC 50 values for inhibition of lipoxygenase activities for Embodiment 1 (luteolin), 2 (chrysoeriol), Comparison 1 (rosemarinic acid) and Comparison 2 (rosemarinic acid methyl ester), together with Comparison 5 (caffeic acid) and 6 (quercetin) measured in the same manner.
- Embodiment 1 lutelion
- IC 50 value of Embodiment 1 is 0.1 M which is very much higher than that of the other compounds, and showed a significant 5-LO inhibitory activity.
- Blood was collected from normal Wistar-King rats (150-400 g) and 0.5 mM of EDTA was added as anti-coagulant. The centrifuged supernatant (1,200 rpm for 10 min.) was again centrifuged at 3,000 rpm for 10 min. The deposited platelets were washed by 25 mM Tris/HCl buffer (pH 7.4) 1 mM EDTA /130 mM NaCl. The suspension of platelets in 1 mM EDTA was homogenized and used as an enzyme solution of 12-lipoxygenase.
- Embodiment 1 to 4 Four to six testing solutions with different levels of concentration were prepared for Embodiment 1 to 4. 0.13 mL of enzyme solution (2 mg protein/mL) and [1- 14 C] arachidonic acid (0.05 Ci) were incubated with a testing solution (0.02 mL) at 37° C. for 5 min.
- the reaction was terminated by adding 0.2 mL of 0.5 N formic acid, and the metabolite was extracted by adding 3 mL of ethyl acetate. Then, the extracted substances were subjected to thin-layer chromatography (TLC) and developed with chloroform/methanol/acetic acid/water (90:8:1:0.8, v/v). The radioactive metabolites of 12 lipoxygenase were detected and quantified using autoradiography. The inhibitory activity of 12-lipoxygenase was measured and expressed as a percentage of the control.
- Embodiments 1 to 4 had significant inhibitory activities against 12-lipoxygenase.
- Embodiment 1 (luteolin) was especially effective, as indicated in FIG. 3 . It showed a very strong inhibitory activity, similar to that against 5-lipoxygenase.
- Embodiment 4 (ethyl acetate fraction) as indicated in FIG. 6, showed a stronger inhibitory activity than Embodiment 3 (ethanolic extraction), again probably because the active compounds of ethanolic extract were concentrated in the ethyl acetate layer.
- Test solutions for Embodiment 1, 3 and 4 were prepared and applied to the right ears of mice (ICR, 5 weeks old, Clair) by adding acetone solution of TPA (0.8 ⁇ g/20 ⁇ l/ear). After 4 hours, the weights of the right and left ears were measured to give the expansion coefficient of dropsical swelling on the right ear against the left ear. The regulating coefficient of inflammation against mice in a control group was also measured. ‘Control’ herein means that the same test was also conducted on a group without applying samples of test solutions under the same condition.
- Embodiment 1 As indicated in Table 2, luteolin (0.3 mg/ear) of Embodiment 1, regulated TPA-induced inflammation on the ear by 100%, Embodiment 3 (ethanolic extraction: 0.5 mg/ear) gave 25% reduction, and Embodiment 4 (ethyl acetate fraction: 1.0 mg/ear) gave 45% reduction, while using the Regulating coefficient of Comparison 7 (NDGA: 0.5 mg/ear) as the substance for inhibition of the enzyme activity of 5-lipoxygenase gave 40% reduction. In other words, Embodiments 1, 3 and 4 had a significant regulating effect against inflammation. Also, Embodiment 1 showed an anti-inflammatory action, which correlated with the concentration of luteolin.
- mice As a sample, 5 g of food containing 1% of Embodiment 4 (ethanolic extraction) was given to the mice for 4 weeks, then TPA acetone solution (0.8 ⁇ g/20 ⁇ l/ear) was applied to the right ears of mice (I.C.R, 5 weeks old/Clair). After 4 hours, the weights of the right and left ears were respectively measured to give the expansion coefficient of inflammation on the right ear against the left ear together with the regulating coefficient of inflammation against mice in a control group. ‘Control’ herein means that the test was conducted on a group without applying samples under the same conditions. The results are shown in Table 2.
- Embodiment 4 As indicated in Table 2, the regulating coefficient of Embodiment 4 (ethanolic extraction: 1% with food) against inflammation was 20%, and oral application also provided a significant anti-inflammatory action. No increases or decreases in weight were found in comparison with the control group.
- Oxazolone solution in ethanol 500 ⁇ g/100 ⁇ l was applied to the abdominal region of mice (I.C.R, 5 weeks old, Clair), that had been shaved by hair clippers under an anesthetic of Nembutal (Immunization).
- oxazolone solution in acetone containing a prescribed amount of the sample being tested (Embodiment 1)
- acetone containing a prescribed amount of the sample being tested
- the weights of the right and left ears were measured to give the expansion coefficient of inflammation on the right ear against the left ear.
- the regulating coefficient of inflammation against mice in a control group was also measured.
- Control herein means that the test was also conducted on a group without applying samples under the same conditions.
- Embodiment 1 showed a higher regulating coefficient for a smaller amount of application against Comparisons 7 to 10.
- luteolin as an active compound has a significant anti-allergic action.
- mice From the beginning of the application until when the weights of ears were measured, 5 g of food mixed with 1% of sample (Embodiment 3) was given to the mice every day. On the 8th day from the beginning of application, immunization was applied to the shaved abdominal region of the mice by an ethanol solution of oxazolone. After 5 days, a solution of oxazolone in acetone was applied to the right ears of the mice (Challenge). After 48 hours, the weights of the right and left ears were measured to give the expansion coefficient of inflammation on the right ear against the left ear. The regulating coefficient of inflammation against mice in a control group was also measured. ‘Control’ herein means that the test was conducted on a group without applying samples under the same conditions. The results are shown in Table 4.
- Embodiment 3 ethanolic extraction: 1% mixed with food
- oxazolone-induced inflammation on the ear by 34%.
- oral application also provided a significant anti-allergic action.
- No increases or decreases in weight of mice in Embodiment 3 was found compared with that of the control group.
Abstract
Novel inhibitors of the enzyme action of lipoxygenase, especially 5-lipoxygenase and 12-lipoxygenase that are derived from the extraction of the seeds of the perilla (crispa) or perilla (frutescens) plant. These inhibitors are suitably extracted from these seeds using alcohol, preferably ethanol, to form an extract or more preferably to further extract the alcoholic extract with ethyl acetate and water to partition the active inhibitors to the ethyl acetate. The particularly preferred inhibitors are luteolin and chrysoeriol.
Description
This invention relates to inhibitors of lipoxygenase.
This invention, in regard to inhibitors of lipoxygenase, is intended for application in medicines regulating or preventing allergic diseases, inflammatory responses or the like. It is intended for use in connection with non-medical supplies, such as cosmetics or food additives and so forth.
Arachidonic acid, an essential fatty acid, exists inside the body as one compound of cell walls. Once arachidonic acid is stimulated, it is separated from phospholipid by the action of phospholipase. The separated arachidonic acid is metabolized by arachidonic acid metabolic enzymes e.g. lipoxygenase, then changed to substances such as prostaglandin (PG) which is associated with inflammatory responses or the like, *thromboxane (TX) which is associated with the formation of thrombus, and leukotriene (LT) and *lipoxyne, which induce allergic reactions. (See FIG. 1).
Thus, arachidonic acid metabolic enzymes create substances which are the source of compounds that are associated with circulatory and allergic diseases, inflammation and so forth. Therefore, in order to regulate or prevent these diseases, it may be effective to inhibit the metabolism of arachidonic acid by enzyme action.
Some typical enzymes relating to the metabolism of arachidonic acid are 5-lipoxygenase, 12-lipoxygenase, and cyclooxynase (COX). 5-Lipoxygenase metabolizes arachidonic acid into 5-hydroxy-6,8,10,14-eicosatetraenoic acid (5-HETE), and induces the formation of leukotriene (LT) which is associated with allergic diseases, inflammatory responses and asthma. Also, 12-lipoxygenase is an enzyme having an arachidonic acid radical that metabolizes into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). This compound is associated with arteriosclerosis, allergic diseases and metastasis of cancer.
Caffeic acid and quercetine or the like are well recognized as natural inhibitors of lipoxygenase. These substances, however, have not come into practical use as enzyme inhibitors. On the other hand, it is known that very few substances show significant inhibitory activity against 12-lipoxygenase. Other polyphenol group members are natural inhibitors of the activity of lipoxygenase. However, the inhibitory activity of these materials is not sufficient to produce satisfactory results in alleviating disease conditions. Further, it is difficult to cost-effectively purify enzyme inhibitors from natural substances.
Perilla (frutescens) seed is a year-round plant belonging to the perilla (crispa) family. It is indigenous to East Asia and is classified as an oil crop. Perilla (crispa) typically has a square-shaped stem, oval-shaped opposed leaves and small white flowers that bloom in the summer season. Its seeds are a little larger than those of perilla (crispa) and are harvested in the autumn.
Oil obtained from perilla (frutescens) seed is known as perilla oil, and is used as an ingredient in food or paint. Also, oil cake is used as fertilizer or feed. It has already been reported that perilla leaves contain physiologically active substances, however, there have been few studies of perilla (frutescens) seed.
As the result of investigations of rosemarinic acid and its methyl ester carried out by the inventors, from among the five substances that inhibit the enzyme activity of lipoxygenase, i.e.: luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester, and apigenin contained in perilla (frutescens) seed, it was found that Japanese Published Examined Patent Application No. 1-121217 has already described the inhibitory activity against 5-lipoxygenase associated with rosemarinic acid or its derivative extracted from fresh leaves of perilla (crispa) family.
Also, Japanese Published Examined Patent Application No. 8-510735 describes the inhibitory activity of apigenin on 5-lipoxygenase.
An imporantant object of this invention is to provide means to significantly inhibit the activities of 5-lipoxygenase and 12-lipoxygenase in relation to the metabolism of arachidonic acid.
Another object of this invention is to extract active inhibitors of lipoxygenase from natural substances that may be used to regulate and prevent allergic diseases, inflammatory responses, circulatory diseases and metastasis of cancer.
Other and additional objects of this invention will become apparent from a consideration of this entire specification, including the drawings hereof and the claims appended hereto.
In accord with and fulfilling these objects, one aspect of this invention is the use of luteolin as an active compound to inhibit the enzyme action of lipoxygenase.
Another feature of this invention is the use of chrysoeriol as an active compound to inhibit the enzyme action of lipoxygenase.
Still another feature of this invention is the compounding of inhibitors of lipoxygenase from alcoholic extracts of perilla (frutescens) seed. These extracts comprise one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin.
A further feature of this invention is that inhibitors of the enzyme action of lipoxygenase can be prepared from the alcoholic extract of perilla (frutescens) seed, including one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester, and apigenin. This alcoholic extract is suitably subjected to partition by ethyl acetate and water (two layers) and the fraction that partitions to the ethyl acetate layer is the desirable inhibitory fraction.
It may be preferable to use defatted perilla (frutescens) seed in place of the aforementioned untreated perilla (frutescens) seed. It is also possible to use perilla (crispa) seed in place of the aforementioned perilla (frutescens) seed.
The inventors have carried out various experiments on extracts of perilla (frutescens) seed, and have thereby discovered that an ethanol extract of perilla (frutescens) seed has a strong inhibitory effect against the enzyme action of 5-lipoxygenase and 12-lipoxygenase. As a result of a close study, the inventors have discovered that the luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and/or apigenin in an alcoholic extract of perilla (frutescens) seed strongly inhibit lipoxygenase enzyme activity. It was found that luteolin especially has an outstanding inhibitory activity against lipoxygenase. The outstanding inhibitory activity of luteolin on lipoxygenase, together with the lesser inhibitory activity of chrysoeriol, has only come to light through the research of these investigations.
The inventors have also found, through various experiments on seeds of other varieties of perilla (crispa) belonging to the same family as perilla (frutescens), that alcoholic extracts of other seeds also contain substances that effectively inhibit the enzyme activity of lipoxygenase, i.e. luteolin, chrysoeriol. rosemarinic acid and rosemarinic acid methyl ester.
It is known that luteolin exists as a glycoside generally in the bean family i.e. digitalis, etc. Also, it is known that luteolin has physiological activity as an anti-oxidant, a hyaluronidase inhibitor, and so forth.
According to this invention, when using luteolin as an active compound for its lipoxygenase inhibitory activity, it can be extracted or purified from the rind of citrus fruits as well as from perilla (frutescens) seed or perilla (crispa) seed and leaves. However, in industrial production, it may be preferable to use perilla (frutescens) seed or perilla (crispa) seed since the leaves contain only small amounts of luteolin.
There are only a few reports of chrysoeriol having physiological activity as an anti-cancer agent or antibiotic. According to this invention, in order to use chrysoeriol as an active compound for its lipoxygenase inhibitory activity, it can be extracted or purified from perilla (frutescens) seed or perilla (crispa) seed and leaves.
It is preferable to use ethanol as solvent/extractant for the extraction of luteolin and chrysoeriol from perilla (frutescens) seed and perilla (crispa) seed. As well as efficiently extracting the effective substances, the use of ethanol allows the extracts to be available in formulations that are useful for both internal and external application. However, when the extract is to be used for external application only, ethyl acetate, acetone, methanol, butanol and so forth can also be used as the extractant.
In the extraction of the chrysoeriol, or luteolin from perilla seed or leaves, as the case may be, it is preferable to use an alcohol concentration between about 70 and 85% (v/v). If the concentration is less than about 70% (v/v), effective substances will not be sufficiently extracted, and if concentration is greater than about 85% (v/v), perilla (frutescens) seed oil easily dissolves into the alcohol. In order to improve the extraction of the active compound from seed, it is preferable to use repeated alcoholic extractions at various levels of concentration.
Inhibitory substances obtained by alcoholic extraction of perilla (frutescens) seed and perilla (crispa) seed normally contain luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin. It is possible that, under different conditions of extraction, some or any of these substances may not be recovered. However, recovery of at least one of these substances is sufficient to inhibit the effects of lipooxygenase.
The reason why alcoholic extracts of perilla (frutescens) seed or perilla (crispa) seed are best partitioned by ethyl acetate and water is that such a subsequent extraction produces a higher concentration of the active compound. A high concentration of luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester concentrates in the layer of ethyl acetate, while non-active compounds such as glycoside, etc., gather in the water layer. Therefore, the active compound can be more efficiently concentrated by using ethyl acetate to capture these effective compounds.
It is possible make inhibiting compositions by directly compounding the aforementioned alcoholic extract and ethyl acetate partitioned compounds as inhibitors of the enzyme activity of lipoxygenase. However it may also be possible to isolate the active compound contained in these extracts. When isolating luteolin, for example, the ethyl acetate layer that contains the luteolin can be subjected to silica gel column chromatography (chloroform:methanol=10:1) and the most active fraction (that is the fraction with the highest concentration of luteolin) is collected. The luteolin can then be separated as an insoluble fraction from the mixed solvent (for example: chloroform:methanol=15:1).
In this invention, alcoholic extraction using defatted perilla (frutescens) seed and perilla (crispa) seed provides a high concentration of active compounds. If the fat of the perilla (frutescens) seed and of the perilla (crispa) seed is extracted by treatment with organic solvents, the extracted oil contains only a small amount of physiologically active compounds, such as luteolin and chrysoeriol, because these active compounds are concentrated in the defatted fraction.
Hexane, for example, is appropriate as an organic solvent for defatting these seeds because the thus extracted oil can be used for food. In fact, the entire extract, oil and hexane or the like, from defatted perilla (frutescens) seed can also be used as an ingredient of foods and so forth. In the case where the substance extracted from defatted perilla (frutescens) seed is to be used for external application only, it would be possible to use not only hexane but also other non-polar solvent. According to this invention, inhibitors of the enzyme activity of lipoxygenase may be used in medical and non-medical applications. Exemplary non-medical applications include, among others: cosmetics, ingredient of foods and so forth. Since inhibitors of lipoxygenase, as described in this invention, exhibit effective inhibitory activity against the role of lipoxygenase in allergy and inflammation, it will also be appropriate to use them in anti-allergic or anti-inflammatory drugs or medicines.
In this invention, inhibitors of the enzyme activity of lipoxygenase may be administered either orally or by other means, such as topically. In the case of oral administration, they can be given in tablet, granule, small grain, or powder form. In the case of non-oral administration, inhibitors of lipoxygenase can be given by injection, intravenous drip, as solid, in suspension, in a viscoelastic fluid i.e. as a suppository that can be absorbed through the mucous membrane, by local topical application to internal or external organic tissue, or by other external administration i.e. intradermal, hypodermic, intramascular and intravenous injection, local topical application, spray, suppository, or injection to the bladder and so forth.
The dose to be administered may fluctuate in accordance with administrative method, seriousness of the condition, age of the patient, condition and general situation of disease, stage of disease and so on. Normally, adults can take about 0.5 to 5,000 mg of active compound a day and children preferably can take about 0.5 to 3,000 mg of active compound a day.
The concentration of inhibitors of lipoxygenase can be varied according to the type of administration. In case of oral administration or absorption through the mucous membrane, the concentration can be approximately 0.3 to 15.0wt %, and in case of non-oral administration, it can be approximately 0.01 to 10 wt %. It should be understood that the above mentioned amount is only exemplary and it is within the scope of this invention to vary the concentration and dosage in accordance with prevailing conditions.
When the inhibitors of lipoxygenase of this invention are to be used for medical or non-medical supplies or cosmetics, they may be used together with various common components of these formulations. These conventional ingredients are exemplified by:
Oils: animal oil, vegetable oil, mineral oil, ester oil, wax oil, silicon oil, higher alcohols, phospholipid, fatty acids, etc.
Surface active agents: anionic, cationic, amphoteric or nonionic surface active agent
Vitamins: vitamin A, vitamin B, folic acid, nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D, vitamin E, ferulic acid, γ-orizanol etc.
Ultraviolet absorbents: p-aminobenzoic acid, anthranil, salicylic acid, coumrin, benzotriazol, tetrazol, imidazoline, purimidine, dioxane, furan, pyrone, camphor, nucleic acid, allantoin and their conductors, amino acid compound, siconin, baicalin, baicalein, berberine, etc.
Anti-oxidants: ester stearate, nordihydroguaseleten acid, dibutyl hydroxytoluene, butyl hydroxyanisole, para hydroxyanisole, gallic acid propyl ester, sesamol, sesamolin, gossypol, etc.
Thickeners: hydxyethyl cellulose, ethyl cellulose, carboxyethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium, hydroxypropyl cellulose, nitrocellulose, polyvinyl alcohol (PVA), polyvinyl alcohol methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylic acid, carboxyvinyl polymer, gum arabic, tragacanth gum, agar-agar, casein, gelatin, pectin, starch, alginic acid and its salts.
Moisture retention agents: propylene glycol, 1,3 butylene glycol, polyethylene glycol, glycerol, chondroitin sulfate, hyaluronic acid, sodium lactate, etc.
Others: lower alcohols, polyhydric alcohols, water-soluble polymers, pH regulators, antiseptics/preservatives, coloring agents, perfumes, refrigerants, stabilizers, animal and vegetable extracts, animal and vegetable proteins, animal and vegetable polysaccharides, animal and vegetable glycoproteins and their decomposition products, metabolites of incubated microorganisms, blood circulation promoters, other anti-inflammatory agents, cell fraction agent amino acids, and their salts, horn-like material dissolution agent, astringents, wound dressing materials, foam fillers, oral agents, deodorants and the like.
The above mentioned materials can be used in medical or non-medical supplies such as cream, ointment, face lotion, body lotion, milky lotion, pack, oil, soap, including medicated soap, cleansing soap, bath aromatics, shampoo, hair conditioner, sprays. Also, they can be used in sanitary products, non-woven fabrics that are useful for tissues, wet wipes and the like, composites for oral use in case of stomatitis and so forth.
In the case where lipoxygenase inhibitors as described in this invention are to be used in food additives, they can be also used in general foodstuffs such as confectionery, noodles, soup, beverages, and health foods including nutritional supplements. For example, inhibitors of lipoxygenase enzyme activity can be used together with powdered cellulose, and can be spray dried or freeze-dried. Such a powder, granule, tablets or solution can easily be mixed with foods.
5-HETE, which is a metabolite of 5-lipoxygenase, produces leukotriene and thereby induces allergic reactions or inflammatory responses. 12-HETE, which is metabolite of arachidonic acid by the action of 12-lipoxygenase, is known to be a physiologically active substance in regard to arteriosclerosis or metastasis of cancer, Therefore, it is possible to regulate or prevent allergic diseases, inflammatory responses, circulatory diseases, metastasis of cancer, or the like by inhibiting these substances selectively.
As described above, according to this invention, it is possible to extract substances that have significant lipoxygenase enzyme action inhibition from natural substances such as perilla (frutescens) seed and other similar materials such that these may be used in food additives in order to effectively regulate allergic reactions and inflammatory responses.
Furthermore, as described herein, inhibitors of lipoxygenase can be obtained from defatted perilla after perilla (frutescens) seed oil for food has been collected. This means that defatted perilla (frutescens) seed can provide another resource.
This invention provides active inhibitors of the enzyme activity of lipoxygenase. These inhibitors are obtained from natural substances and have significant inhibitory activities against both 5-lipoxygenase and 12-lipoxygenase. These inhibitors are available to regulate or prevent allergic reactions, inflammatory responses, circulatory problems and metastasis of cancer. A feature of the lipoxygenase inhibitors as described in this invention is that at least one of luteolin or chrysoeriol are used as the active compound.
Another feature of this invention is that lipoxygenase inhibitors of this invention is the recovery thereof from alcoholic extract of perilla (frutescens) seed containing one or more compounds from among luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin. A further feature is that the aforementioned alcoholic extract is partitioned with ethyl acetate and water, and especially effective inhibitors are obtained from the ethyl acetate layer. Defatted perilla (frutescens) seed may preferably be substituted for whole perilla (frutescens) seed. Perilla (crispa) seed can be substituted for or used with the aforementioned perilla (frutescens) seed.
FIG. 1 shows the physiological action of arachidonic acid metabolic enzyme.
FIG. 2 is a flow chart to describe the method for isolation of inhibitors of lipoxygenase in accordance with the examples in this invention.
FIG. 3 shows the correlation between the concentration of isolated luteolin and inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the embodiments in this invention.
FIG. 4 shows the correlation between the concentration of isolated chrysoeriol and inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the embodiments of this invention.
FIG. 5 shows the correlation between the concentration of ethanolic extraction and ethyl acetate fraction, and the inhibition of 5-lipoxygenase in accordance with the embodiments in this invention.
FIG. 6 shows the correlation between the concentration of ethanolic extraction and ethyl acetate fraction, and the inhibition of 12-lipoxygenase in accordance with the embodiments in this invention.
FIG. 7 shows the correlation between the concentration of isolated rosemarinic acid and the inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the comparisons in this invention.
FIG. 8 shows the correlation between the concentration of isolated rosemarinic acid methyl ester and the inhibition of 5-lipoxygenase and 12-lipoxygenase in accordance with the comparisons in this invention.
Isolation of Lipoxygenase Inhibitors
As described in FIG. 2, crushed perilla (frutescens) seed was extracted (defatted) with hexane, and, after separation of the fat fraction, the residue of defatted perilla (frutescens) seed was then extracted by refluxing with 80% aqueous ethanol. The concentrated ethanolic extract was partitioned by hexane and 80% aqueous methanol. The 80% aqueous methanolic layer was evaporated to dryness and then partitioned between ethyl acetate and water. After separating the ethyl acetate layer from the water layer, some of the ethyl acetate solvent was distilled off, and a fraction of product in a smaller quantity of ethyl acetate was thus obtained.
The remaining ethyl acetate fraction was subjected to silica gel column chromatography (using a solvent comprising chloroform:methanol=10:1) to give further separate fractions.
The pure luteolin and pure chrysoeriol isolated from perilla (frutescens) seed in this manner are respectively designated Embodiment 1 and 2.
The ethanolic extraction (as illustrated in FIG. 2) obtained by evaporation of solvent was designated Embodiment 3, and the ethyl acetate layer obtained by partitioning the ethanolic extraction fraction was evaporated with the solvent, and then the fraction in the ethyl acetate was designated Embodiment 4.
Test of 5-Lipoxygenase Inhibitory Activity
A test of the substances' effectiveness as inhibitors of 5-lipoxygenase was conducted for the above mentioned Embodiment 1 to 4.
(1) Formation of Enzyme Solution of 5-Lipoxygenase
Peritoneal polymorphonuclear leukocytes (PMNL) were prepared from normal Wistar-King rats (150-400 g) injected intraperitoneally with 5% (w/v) glycogen (20 mL/kg). A suspension in potassium phosphate buffer (50 mM, pH 7.4) was homogenized and centrifuged. The supernatant was used as the enzyme solution of 5-lipoxygenase.
(2) Measurement of Inhibitory Activity Against 5-Lipoxygenase
Four to six testing solutions with different levels of concentrations were prepared for Embodiments 1 to 4. 0.02 mL Of enzyme solution (2 mg protein/mL) and [1l-14C] arachidonic acid (0.05 Ci) were incubated with each testing solution (0.02 mL) and Ca2+ and ATP at 37° C. for 5 min.
After incubation at 37° C. for 5 min, the reaction was terminated by adding 0.2 mL of 0.5 N formic acid, and the metabolite was extracted by 3 mL of ethyl acetate.
Then the extracted solution was subjected to thin-layer chromatography (TLC) and developed with ether/petroleum ether/acetic acid (50:50:1, v/v) at 4° C. The radioactive metabolites generated by 5-lipoxygenase were detected and quantified using autoradiography. The inhibitory activity on 5-lipoxygenase was measured and expressed as a percentage of the control.
For comparison, inhibition of 5-lipoxygenase activity was measured in the same manner, for rosemarinic acid (Comparison 1) obtained by purifying fraction 3 as indicated in FIG. 2, rosemarinic acid methyl ester (Comparison 2), hexane fraction (Comparison 3) obtained by evaporating hexane layer with solvent, and water fraction (Comparison 4) obtained by evaporating the water layer also with the solvent. The results are shown in FIGS. 3 to 5, 7 and 8.
As a result, Embodiments 1 to 4 showed significant inhibitory activities against 5-lipoxygenase. Embodiment 1 (luteolin) especially, as indicated in FIG. 3, showed a stronger inhibitory activity than that of Comparison 1 (rosemarinic acid) and Comparison 2 (rosemarinic acid methyl ester) which had already been reported to have 5-lipoxygenase inhibitory action.
Embodiment 4 (ethyl acetate fraction) showed even more significant inhibitory activity than that of Embodiment 3 (ethanolic extraction). The reason for this phenomenon seems to be that active compounds are concentrated in the ethyl acetate layer by eliminating less active water and oil soluble substances from the ethanolic extract. Also, the inhibitory activities of Embodiment 3 (ethanolic extraction) and 4 (ethyl acetate fraction) seem to be correlated with their luteolin content.
Table 1 shows the IC50 values for inhibition of lipoxygenase activities for Embodiment 1 (luteolin), 2 (chrysoeriol), Comparison 1 (rosemarinic acid) and Comparison 2 (rosemarinic acid methyl ester), together with Comparison 5 (caffeic acid) and 6 (quercetin) measured in the same manner.
TABLE 1 |
IC50 Values for Inhibition of 5-Lipoxygenase |
Activity by Phenolic Compounds |
IC50 (M) | ||||
Classification | Compound | 5- | ||
Embodiment |
1 | luteolin | 0.1 | |
|
chrysoeriol | 22.0 | |
|
rosemarinic acid | 6.2 | |
|
rosemarinic acid methyl ester | 0.6 | |
|
caffeic acid | 72.0 | |
Comparison 6 | quercetin | 0.2 | |
As shown by the comparative data reported in Table 1, the IC50 value of Embodiment 1 (lutelion) is 0.1 M which is very much higher than that of the other compounds, and showed a significant 5-LO inhibitory activity.
Test of 12-Lipoxygenase Inhibitory Activity
A test of the substances' effectiveness as inhibitors of the enzyme activity of 12-lipoxygenase was conducted for Embodiment 1 to 4.
(1) Formation of Enzyme Solution of 12-Lipoxygenase
Blood was collected from normal Wistar-King rats (150-400 g) and 0.5 mM of EDTA was added as anti-coagulant. The centrifuged supernatant (1,200 rpm for 10 min.) was again centrifuged at 3,000 rpm for 10 min. The deposited platelets were washed by 25 mM Tris/HCl buffer (pH 7.4) 1 mM EDTA /130 mM NaCl. The suspension of platelets in 1 mM EDTA was homogenized and used as an enzyme solution of 12-lipoxygenase.
(2) Measurement of Inhibitory Activity of 12-Lipoxygenase
Four to six testing solutions with different levels of concentration were prepared for Embodiment 1 to 4. 0.13 mL of enzyme solution (2 mg protein/mL) and [1-14C] arachidonic acid (0.05 Ci) were incubated with a testing solution (0.02 mL) at 37° C. for 5 min.
After incubation at 37° C. for 5 min., the reaction was terminated by adding 0.2 mL of 0.5 N formic acid, and the metabolite was extracted by adding 3 mL of ethyl acetate. Then, the extracted substances were subjected to thin-layer chromatography (TLC) and developed with chloroform/methanol/acetic acid/water (90:8:1:0.8, v/v). The radioactive metabolites of 12 lipoxygenase were detected and quantified using autoradiography. The inhibitory activity of 12-lipoxygenase was measured and expressed as a percentage of the control.
As a comparison, inhibition of 12-lipoxygenase enzyme activity was measured in the same manner, for rosemarinic acid (Comparison 1) obtained by purifying fraction 3 as indicated in FIG. 2, rosemarinic acid methyl ester (Comparison 2), hexane fraction (Comparison 3) obtained by evaporating hexane layer with solvent, and water fraction (Comparison 4) obtained by evaporating water layer also with solvent. The results are shown in FIGS. 3, 4 and 6 to 8.
As shown, Embodiments 1 to 4 had significant inhibitory activities against 12-lipoxygenase. Embodiment 1 (luteolin) was especially effective, as indicated in FIG. 3. It showed a very strong inhibitory activity, similar to that against 5-lipoxygenase.
Embodiment 4 (ethyl acetate fraction) as indicated in FIG. 6, showed a stronger inhibitory activity than Embodiment 3 (ethanolic extraction), again probably because the active compounds of ethanolic extract were concentrated in the ethyl acetate layer.
As described in this section, a test for regulating action against TPA-induced inflammation to the ears of mice was conducted.
(1) Application to the Skin
Test solutions for Embodiment 1, 3 and 4 were prepared and applied to the right ears of mice (ICR, 5 weeks old, Clair) by adding acetone solution of TPA (0.8 μg/20 μl/ear). After 4 hours, the weights of the right and left ears were measured to give the expansion coefficient of dropsical swelling on the right ear against the left ear. The regulating coefficient of inflammation against mice in a control group was also measured. ‘Control’ herein means that the same test was also conducted on a group without applying samples of test solutions under the same condition.
Also, as a comparison, data on NDGA (Comparison 7) which has already been reported, are listed in Table 2.
TABLE 2 | ||||
Regulating | ||||
Class. | Compound | Application | Amount | Coefficient(%) |
|
luteolin | skin | 0.15 mg/ear | 85 |
|
luteolin | skin | 0.3 mg/ |
100 |
|
ethanolic extraction | skin | 0.5 mg/ear | 25 |
|
ethyl acetate fraction | skin | 1.0 mg/ear | 45 |
Comparison 7 | NDGA | skin | 0.5 mg/ear | 40 |
|
ethanolic extraction | oral | 1.0% mixed w/food | 20 |
Note: Comparison 7 | ||||
Source: “I-1 Evaluation of anti-inflammatory medicine and anti-allergic medicine by animal experiment” of “Vol. 12 - Inflammation and Allergy” compiled by Prof. Kazuo Ouchi, Pharmaceutical Department of Tohoku Univ. & Published by Hirokawa Shoten |
As indicated in Table 2, luteolin (0.3 mg/ear) of Embodiment 1, regulated TPA-induced inflammation on the ear by 100%, Embodiment 3 (ethanolic extraction: 0.5 mg/ear) gave 25% reduction, and Embodiment 4 (ethyl acetate fraction: 1.0 mg/ear) gave 45% reduction, while using the Regulating coefficient of Comparison 7 (NDGA: 0.5 mg/ear) as the substance for inhibition of the enzyme activity of 5-lipoxygenase gave 40% reduction. In other words, Embodiments 1, 3 and 4 had a significant regulating effect against inflammation. Also, Embodiment 1 showed an anti-inflammatory action, which correlated with the concentration of luteolin.
(2) Oral Application
As a sample, 5 g of food containing 1% of Embodiment 4 (ethanolic extraction) was given to the mice for 4 weeks, then TPA acetone solution (0.8 μg/20 μl/ear) was applied to the right ears of mice (I.C.R, 5 weeks old/Clair). After 4 hours, the weights of the right and left ears were respectively measured to give the expansion coefficient of inflammation on the right ear against the left ear together with the regulating coefficient of inflammation against mice in a control group. ‘Control’ herein means that the test was conducted on a group without applying samples under the same conditions. The results are shown in Table 2.
As indicated in Table 2, the regulating coefficient of Embodiment 4 (ethanolic extraction: 1% with food) against inflammation was 20%, and oral application also provided a significant anti-inflammatory action. No increases or decreases in weight were found in comparison with the control group.
Allergy Control Test Against Oxazolone-Induced Inflammation in the Ears of Mice
As described in this section, an allergy control test of oxazolone-induced inflammation to the ears of mice was conducted.
(1) Application to the Skin
Oxazolone solution in ethanol (500 μg/100 μl) was applied to the abdominal region of mice (I.C.R, 5 weeks old, Clair), that had been shaved by hair clippers under an anesthetic of Nembutal (Immunization).
After 5 days of immunization, oxazolone solution in acetone, containing a prescribed amount of the sample being tested (Embodiment 1), was applied to the right ears of the mice (Challenge). After 48 hours, the weights of the right and left ears were measured to give the expansion coefficient of inflammation on the right ear against the left ear. The regulating coefficient of inflammation against mice in a control group was also measured. ‘Control’ herein means that the test was also conducted on a group without applying samples under the same conditions.
Also, as a comparison, data on NDGA (Comparison 7), ketoprophen (Comparison 8), phenidone (Comparison 9) and mepiramine (Comparison 10) which had already been reported, were listed in Table 3.
TABLE 3 | ||||
Regulating | ||||
Class. | Compound | Application | Amount | Coefficient(%) |
|
luteolin | skin | 0.3 mg/ear | 52 |
Comparison 7 | NDGA | skin | 1.0 mg/ear | 38 |
Comparison 8 | ketoprophene | skin | 1.0 mg/ear | 32 |
Comparison 9 | phenidone | skin | 1.0 mg/ear | 44 |
|
mepiramin | skin | 1.0 mg/ear | 38 |
|
ethanolic extraction | oral | 1.0% mixed w/food | 34 |
Note: Comparison 7 to 10 | ||||
Source: “I-1 Evaluation of anti-inflammatory medicine and anti-allergic medicine by animal experiment” of “Vol. 12 - Inflammation and Allergy” compiled by Prof. Kazuo Ouchi, Pharmaceutical Department of Tohoku Univ. & Published by Hirokawa Shoten |
As indicated in Table 3, Embodiment 1 showed a higher regulating coefficient for a smaller amount of application against Comparisons 7 to 10. In other words, luteolin as an active compound has a significant anti-allergic action.
(2) Oral Application
From the beginning of the application until when the weights of ears were measured, 5 g of food mixed with 1% of sample (Embodiment 3) was given to the mice every day. On the 8th day from the beginning of application, immunization was applied to the shaved abdominal region of the mice by an ethanol solution of oxazolone. After 5 days, a solution of oxazolone in acetone was applied to the right ears of the mice (Challenge). After 48 hours, the weights of the right and left ears were measured to give the expansion coefficient of inflammation on the right ear against the left ear. The regulating coefficient of inflammation against mice in a control group was also measured. ‘Control’ herein means that the test was conducted on a group without applying samples under the same conditions. The results are shown in Table 4.
As indicated in Table 4, Embodiment 3 (ethanolic extraction: 1% mixed with food) regulated oxazolone-induced inflammation on the ear by 34%. In other words, oral application also provided a significant anti-allergic action. No increases or decreases in weight of mice in Embodiment 3 was found compared with that of the control group.
Claims (24)
1. A composition comprising a sufficient amount of an ethanol extract of perilla seed, comprising at least one compound selected from the group consisting of luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin, effective to inhibit the enzyme action of lipoxygenase.
2. The composition as claimed in claim 1 wherein said ethanol extract is derived from defatted perilla (crispa) seed.
3. A method of combating an allergic reaction and inflammatory responses which comprises administering, to an animal having an allergic reaction, an inhibitor of the enzyme action of lipoxygenase comprising a composition as claimed in claim 1.
4. A method of combating an allergic reaction and inflammatory responses as claimed in claim 3 wherein said composition comprises luteolin.
5. A method of combating an allergic reaction and inflammatory responses as claimed in claim 3 wherein said composition comprises chrysoeriol.
6. A comestible comprising the composition claimed in claim 1.
7. A cosmetic comprising the composition claimed in claim 1.
8. A method of combating allergic reaction and inflammatory responses which comprises administering, to an animal having an allergic reaction, an inhibitor of the enzyme action of lipoxygenase comprising an ethanol extract of perilla seed, comprising an amount of at least one compound selected from the group consisting of luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin, effective to inhibit the enzyme action of lipoxygenase.
9. The method of combating allergic reaction and inflammatory responses as claimed in claim 8 wherein said ethanol extract is derived from the species perilla (frutescens) seed.
10. The method of combating allergic reaction and inflammatory responses as claimed in claim 5 wherein said ethanol extract is derived from the species perilla (crispa) seed.
11. The method of combating allergic reaction and inflammatory responses as claimed in claim 5 wherein said ethanol extract is derived from defatted species perilla (crispa) seed.
12. A method of combating allergic reaction and inflammatory responses which comprises administering, to an animal having an allergic reaction, an inhibitor of the enzyme action of lipoxygenase comprising an ethyl acetate extract of perilla seeds comprising an amount of at least one compound selected from the group consisting of luteolin, chrysoeriol, rosemarinic acid, rosemarinic acid methyl ester and apigenin, effective to inhibit the enzyme action of lipoxygenase.
13. The method of combating allergic reaction and inflammatory responses as claimed in claim 12 wherein said ethyl acetate extract is derived from the species perilla (frutescens) seed.
14. The method of combating allergic reaction and inflammatory responses as claimed in claim 12 wherein said ethyl acetate extract is derived from the species perilla (crispa) seed.
15. The method of combating allergic reaction and inflammatory responses as claimed in claim 12 wherein said ethyl acetate extract is derived from defatted species perilla (crispa) seed.
16. A method of inhibiting the enzyme action of lipoxygenase that comprises administering to an animal in need thereof an effective amount of an ethanol or ethyl acetate extract of perilla seed comprising at least luteolin and chrysoeriol.
17. A composition comprising an ethanol extract of perilla seed.
18. The composition as claimed in claim 17 comprising luteolin.
19. The composition as claimed in claim 18 wherein said perilla seed comprises perilla (frutescens) seed.
20. The composition as claimed in claim 18 wherein said perilla seed comprises perilla (crispa) seed.
21. The composition as claimed in claim 17 comprising chrysoeriol.
22. The composition as claimed in claim 19 wherein said perilla seed comprises perilla (frutescens) seed.
23. The composition as claimed in claim 19 wherein said perilla seed comprises perilla (crispa) seed.
24. A pharmaceutical composition comprising a composition as claimed in claim 17 admixed with a pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9-105959 | 1997-04-23 | ||
JP10595997A JP4231559B2 (en) | 1997-04-23 | 1997-04-23 | Lipoxygenase inhibitor |
Publications (1)
Publication Number | Publication Date |
---|---|
US6217875B1 true US6217875B1 (en) | 2001-04-17 |
Family
ID=14421355
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/053,064 Expired - Lifetime US6217875B1 (en) | 1997-04-23 | 1998-04-01 | Inhibitors of lipoxygenase |
Country Status (2)
Country | Link |
---|---|
US (1) | US6217875B1 (en) |
JP (1) | JP4231559B2 (en) |
Cited By (32)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010024664A1 (en) * | 1999-03-19 | 2001-09-27 | Obukowicz Mark G. | Selective COX-2 inhibition from edible plant extracts |
US20020122836A1 (en) * | 2000-12-15 | 2002-09-05 | Pharmacia Corporation | Selective COX-2 inhibition from non-edible plant extracts |
US20020136784A1 (en) * | 2000-12-15 | 2002-09-26 | Pharmacia Corporation | Selective COX-2 inhibition from plant extracts |
US6599539B1 (en) * | 2002-07-31 | 2003-07-29 | Poulenger Usa Inc. | Perilla seed pesticide |
US20030158237A1 (en) * | 2001-09-04 | 2003-08-21 | Colba R & D Inc. | Combination of antioxidant substances for the treatment of alzheimer's disease |
US20030215531A1 (en) * | 2000-09-28 | 2003-11-20 | Martin Stogniew | Compositions and methods of use for extracts of Rutaceae plants |
US20030216481A1 (en) * | 2002-04-30 | 2003-11-20 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-ring flavonoids and flavans as a therapeutic agent |
US20040062823A1 (en) * | 2001-12-13 | 2004-04-01 | Obukowicz Mark G. | Selective cox-2 inhibition from non-edible plant extracts |
US20040191327A1 (en) * | 2003-03-24 | 2004-09-30 | Council Of Scientific And Industrial Research | Method of treating and/or preventing asthma using natural compound luteolin |
US20040220119A1 (en) * | 2003-04-04 | 2004-11-04 | Unigen Pharmaceuticals, Inc. | Formulation of dual cycloxygenase (COX) and lipoxygenase (LOX) inhibitors for mammal skin care |
US20050096281A1 (en) * | 2002-03-01 | 2005-05-05 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-Ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments |
US20050112699A1 (en) * | 2003-07-24 | 2005-05-26 | Peter Sutovsky | Methods and compositions for evaluation and modulation of fertility |
US20060078630A1 (en) * | 2003-02-26 | 2006-04-13 | Schempp Christoph M | Method for the production of flavonoid-containing compositions and use thereof |
US20060079467A1 (en) * | 2002-04-30 | 2006-04-13 | Unigen Pharmaceuticals, Inc. | Formulation of dual eicosanoid system and cytokine system inhibitors for use in the prevention and treatment of oral diseases and conditions |
US20060134236A1 (en) * | 2004-02-05 | 2006-06-22 | Access Business Group International Llc | Anti-allergy composition and related method |
US7192611B2 (en) | 2002-03-01 | 2007-03-20 | Unigen Pharmaceuticals, Inc. | Identification of Free-B-Ring flavonoids as potent COX-2 inhibitors |
US20070135359A1 (en) * | 2003-02-28 | 2007-06-14 | Unigen Pharmaceuticals, Inc. | Identification of Free-B-Ring Flavonoids as Potent COX-2 Inhibitors |
US20080096827A1 (en) * | 2002-04-30 | 2008-04-24 | Unigen Pharmaceuticals, Inc. | Formulation Of A Mixture Of Free-B-Ring Flavonoids And Flavans For Use In The Prevention And Treatment Of Cognitive Decline And Age-Related Memory Impairments |
CN100438867C (en) * | 2004-03-05 | 2008-12-03 | 惠汝太 | Application of flavone monomer in antivirus drug preparing process |
CN100569241C (en) * | 2007-01-17 | 2009-12-16 | 北京天川军威医药技术开发有限公司 | Celery seed acetic acid ethyl ester extract and uses thereof |
CN103099845A (en) * | 2013-02-25 | 2013-05-15 | 中国农业科学院农业质量标准与检测技术研究所 | Method for extracting perillaseed hydrophilic chemical component from perillaseed oil residue |
WO2013136070A1 (en) * | 2012-03-13 | 2013-09-19 | University Court Of The University Of Dundee | A sik inhibitor for use in a method of treating an inflammatory and/or immune disorder |
US8568799B2 (en) | 2002-03-22 | 2013-10-29 | Unigen, Inc. | Isolation of a dual COX-2 and 5-lipoxygenase inhibitor from acacia |
CN106333994A (en) * | 2015-07-10 | 2017-01-18 | 财团法人医药工业技术发展中心 | Perilla seed extract and pharmacological effect thereof |
US10266488B2 (en) | 2013-10-10 | 2019-04-23 | Eastern Virginia Medical School | 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase |
US10265321B2 (en) | 2014-08-08 | 2019-04-23 | Dana-Farber Cancer Institute, Inc. | Uses of salt-inducible kinase (SIK) inhibitors |
US10287268B2 (en) | 2014-07-21 | 2019-05-14 | Dana-Farber Cancer Institute, Inc. | Imidazolyl kinase inhibitors and uses thereof |
US10457691B2 (en) | 2014-07-21 | 2019-10-29 | Dana-Farber Cancer Institute, Inc. | Macrocyclic kinase inhibitors and uses thereof |
EP3791880A1 (en) | 2009-04-29 | 2021-03-17 | Amarin Pharmaceuticals Ireland Limited | Pharmaceutical compositions comprising epa |
US10954242B2 (en) | 2016-07-05 | 2021-03-23 | The Broad Institute, Inc. | Bicyclic urea kinase inhibitors and uses thereof |
US11241435B2 (en) | 2016-09-16 | 2022-02-08 | The General Hospital Corporation | Uses of salt-inducible kinase (SIK) inhibitors for treating osteoporosis |
US11285158B2 (en) | 2017-02-28 | 2022-03-29 | The General Hospital Corporation | Uses of pyrimidopyrimidinones as SIK inhibitors |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001114686A (en) * | 1999-10-15 | 2001-04-24 | Sunstar Inc | Th2 CYTOKININ EXPRESSION SUPPRESSANT |
JP2002212026A (en) * | 2001-01-18 | 2002-07-31 | Oriza Yuka Kk | Skin care preparation for beautifying skin |
JP4441177B2 (en) * | 2001-02-01 | 2010-03-31 | 明治製菓株式会社 | Process for producing phenolic-containing Labiatae plant extract and use thereof |
KR100447948B1 (en) * | 2001-02-27 | 2004-09-08 | 한국생명공학연구원 | Agastache rugosa Extract for anti-inflammatory activity and anti-atherogenic activity |
JP4992008B2 (en) * | 2003-08-29 | 2012-08-08 | 独立行政法人産業技術総合研究所 | Endothelin-1 production inhibitor |
JP2005089385A (en) * | 2003-09-18 | 2005-04-07 | Ryukyu Bio Resource Kaihatsu:Kk | Lipoxygenase inhibitor |
JP4377728B2 (en) * | 2004-03-25 | 2009-12-02 | 明治製菓株式会社 | Novel rosmarinic acid derivatives with anti-inflammatory activity |
WO2006105725A1 (en) * | 2005-04-05 | 2006-10-12 | Shanghai Gloriayx Biopharmaceuticals Co., Ltd | A combination of luteolin and one of pt chemotherapeutics |
AU2006253198B2 (en) | 2005-05-30 | 2010-05-06 | Korea Research Institute Of Bioscience And Biotechnology | Pharmaceutical composition comprising an extract of Pseudolysimachion longifolium and the catalpol derivatives isolated therefrom having antiinflammatory, antiallergic and antiasthmatic activity |
US8455541B2 (en) | 2005-05-30 | 2013-06-04 | Korea Research Institute Of Bioscience And Biotechnology | Pharmaceutical composition comprising an extract of pseudolysimachion longifolium and the catalpol derivatives isolated therefrom having antiinflammatory, antiallergic and antiasthmatic activity |
JP4969802B2 (en) * | 2005-06-17 | 2012-07-04 | 株式会社琉球バイオリソース開発 | Medicinal composition containing Japanese algae and / or a fermented product of Nigana |
JP2007106718A (en) * | 2005-10-14 | 2007-04-26 | Sunstar Inc | Adiponectin secretion promoter and oral composition containing the adiponectin secretion promotor |
JP2007314472A (en) * | 2006-05-26 | 2007-12-06 | Oriza Yuka Kk | Body odor inhibiting agent |
FR2984730A1 (en) * | 2011-12-22 | 2013-06-28 | Diverchim | NEW ANTI-AGE AND DEPIGMENTING COSMETIC COMPOSITIONS |
JP6375087B2 (en) * | 2013-03-04 | 2018-08-15 | 共栄化学工業株式会社 | Cosmetics |
CN116999484A (en) * | 2023-10-07 | 2023-11-07 | 云南英格生物技术有限公司 | Application of perilla seed oil meal extract in preparation of 5 alpha-reductase inhibitor |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4329361A (en) * | 1979-12-22 | 1982-05-11 | Nattermann & Cie Gmbh | Use of rosmarinic acid in the treatment of inflammations and pharmaceutical products used therein |
JPS595110A (en) * | 1982-06-30 | 1984-01-12 | Lion Corp | Composition for oral cavity |
US4708964A (en) * | 1984-02-09 | 1987-11-24 | Chemex Pharmaceuticals | Lipoxygenase inhibitors |
US5043323A (en) * | 1987-01-14 | 1991-08-27 | Indena S.P.A. | Complex compounds of bioflavonoids with phospholipids, their preparation and use, and pharmaceutical and cosmetic compositions containing them |
WO1994027563A1 (en) * | 1993-05-28 | 1994-12-08 | Handelman, Joseph, H. | Inhibition of hair growth |
US5858371A (en) * | 1997-02-05 | 1999-01-12 | Panacea Biotech Limited | Pharmaceutical composition for the control and treatment of anorectal and colonic diseases |
-
1997
- 1997-04-23 JP JP10595997A patent/JP4231559B2/en not_active Expired - Lifetime
-
1998
- 1998-04-01 US US09/053,064 patent/US6217875B1/en not_active Expired - Lifetime
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4329361A (en) * | 1979-12-22 | 1982-05-11 | Nattermann & Cie Gmbh | Use of rosmarinic acid in the treatment of inflammations and pharmaceutical products used therein |
JPS595110A (en) * | 1982-06-30 | 1984-01-12 | Lion Corp | Composition for oral cavity |
US4708964A (en) * | 1984-02-09 | 1987-11-24 | Chemex Pharmaceuticals | Lipoxygenase inhibitors |
US5043323A (en) * | 1987-01-14 | 1991-08-27 | Indena S.P.A. | Complex compounds of bioflavonoids with phospholipids, their preparation and use, and pharmaceutical and cosmetic compositions containing them |
WO1994027563A1 (en) * | 1993-05-28 | 1994-12-08 | Handelman, Joseph, H. | Inhibition of hair growth |
US5858371A (en) * | 1997-02-05 | 1999-01-12 | Panacea Biotech Limited | Pharmaceutical composition for the control and treatment of anorectal and colonic diseases |
Non-Patent Citations (25)
Title |
---|
Baumann, J. et al., Flavonoids and Related Compounds as Inhibitors of Arachidonic Acid Peroxidation, Prostaglandins, vol. 20(4), p. 627-639, Oct. 1980. |
Baumann, J. et al., Prostaglandins, vol. 20(4), p. 627-639, Oct. 1980.* |
Biswas, K.M. et al., Indian J. Chem., vol. 15B(4), p. 396-397, Apr. 1977.* |
Biswas, K.M. et al., Isolation of Chrysoeriol 7-O-beta-D -Glucopyrano-Sidyl (2->1)-D-Apiofuranoside from the Leaves of Dalbergia Volubilis, Indian J. Chem., vol. 15B(4), p. 396-397, Apr. 1977. |
Biswas, K.M. et al., Isolation of Chrysoeriol 7-O-β-D -Glucopyrano-Sidyl (2→1)-D-Apiofuranoside from the Leaves of Dalbergia Volubilis, Indian J. Chem., vol. 15B(4), p. 396-397, Apr. 1977. |
Flamini, G. et al., Phenolic Compounds from Santolina Pinnata, Planta Med., vol. 60(1), p. 97, 1994. |
Flamini, G. et al., Planta Med., vol. 60(1), p. 97, 1994.* |
Gerritsen, M.E. et al., Amer. J. of Pathology, vol. 147(2), p. 278-292, Aug. 1995.* |
Gerritsen, M.E., et al., Flavoniods Inhibit Cytokine-Induced Endothelial Cell Adhesion Protein Gene Expression, Amer. J. of Pathology, vol. 147(2), p. 278-292, Aug. 1985. |
Hsieh, R.J. et al., Lipids, vol. 23(4), p. 322-326, 1988.* |
Hsieh, R.J. et al., Relative Inhibitory Potencies of Flavonoids on 12-Lipoxygenase of Fish Gill, Lipids, vol. 23(4), p. 322-326, 1988. |
Ishikura, Agric. Biol. Chem. 45(8): 1855-1860 (1981).* |
Lyckander, I.M. et al., Lipophilic Flavonoids from Orthosiphon Spicatus as Inhibitors of 15-Lipoxygenase, Acta. Pharm. Nord., vol. 4(3), p. 159-166, 1992. |
Lyckander, I.M.et al., Acta. Pharm. Nord., vol. 4(3), p. 159-166, 1992.* |
Robak, J. et al., Pol. J. Pharmacol. Pharm., vol. 40(5), p. 451-458, 1988.* |
Robak, J. et al., Screening of the Influence of Flavonoids on Liboxygenase and Cyclooxygenase Activity, as well as on Nonenzymic Lipid Oxidation, Pol. J. Pharmacol. Pharm., vol. 40(5), p. 451-458, 1988. |
Silvan, A.M. et al., Effects of Compounds Extracted from Santolina Oblongifolia on TXB2 Relase in Human Platelets, Inflammopharmacology, vol. 6(3), p. 255-263, 1998. |
Silvan, A.M.et al., Inflammopharmacology, vol. 6(3), p. 255-263, 1998.* |
Welton, A.F. et al., Effect of Flovonoids on Arachidonic Acid Metabolism, Prog. Clin. Biol. Res., vol. 213, p. 231-242, 1986. |
Welton, A.F. et al., Prog. Clin. Biol. Res., vol. 213, p. 231-242, 1986.* |
Williams, C.A. et al., A Biologically Active Lipophilic Flavonol from Tanacetum Parthenium, Phytochemistry, vol. 38(1), p. 267-270, 1995. |
Williams, C.A. et al., Phytochemistry, vol. 38(1), p. 267-270, 1995.* |
Yamamoto, Hiroyo et al., Inhibitory Activity of Arachidonic Acid Metabolic Enzyme (In Vitro), Apr. 2, 1997, (Inventor's paper in Japanese with translation. |
Zarga, M.A. et al., Chemical Constituents of Artemisia Arborescens and the Effect of the Aqueous Extract of Rat Isolated Smooth Muscle, Planta Med., vol. 61(3), p. 242-245, 1995. |
Zarga, M.A. et al., Planta Med., vol. 61(3), p. 242-245, 1995.* |
Cited By (70)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20010024664A1 (en) * | 1999-03-19 | 2001-09-27 | Obukowicz Mark G. | Selective COX-2 inhibition from edible plant extracts |
US20040185122A1 (en) * | 1999-03-19 | 2004-09-23 | Pharmacia Corporation | Selective COX-2 inhibition from edible plant extracts |
US20030215531A1 (en) * | 2000-09-28 | 2003-11-20 | Martin Stogniew | Compositions and methods of use for extracts of Rutaceae plants |
WO2002047708A2 (en) * | 2000-12-15 | 2002-06-20 | Pharmacia Corporation | Selective cox-2 inhibition from edible plant extracts |
US20020122836A1 (en) * | 2000-12-15 | 2002-09-05 | Pharmacia Corporation | Selective COX-2 inhibition from non-edible plant extracts |
US20020136784A1 (en) * | 2000-12-15 | 2002-09-26 | Pharmacia Corporation | Selective COX-2 inhibition from plant extracts |
US20040197429A1 (en) * | 2000-12-15 | 2004-10-07 | Pharmacia Corporation | Selective COX-2 inhibition from plant extracts |
WO2002047708A3 (en) * | 2000-12-15 | 2003-12-31 | Pharmacia Corp | Selective cox-2 inhibition from edible plant extracts |
US20040052870A1 (en) * | 2000-12-15 | 2004-03-18 | Obukowicz Mark G. | Selective cox-2 inhibition from edible plant extracts |
US20030158237A1 (en) * | 2001-09-04 | 2003-08-21 | Colba R & D Inc. | Combination of antioxidant substances for the treatment of alzheimer's disease |
US20040062823A1 (en) * | 2001-12-13 | 2004-04-01 | Obukowicz Mark G. | Selective cox-2 inhibition from non-edible plant extracts |
US8535735B2 (en) | 2002-03-01 | 2013-09-17 | Unigen, Inc. | Identification of free-B-ring flavonoids as potent COX-2 inhibitors |
US9061039B2 (en) | 2002-03-01 | 2015-06-23 | Unigen, Inc. | Identification of Free-B-Ring flavonoids as potent COX-2 inhibitors |
US7192611B2 (en) | 2002-03-01 | 2007-03-20 | Unigen Pharmaceuticals, Inc. | Identification of Free-B-Ring flavonoids as potent COX-2 inhibitors |
US20050096281A1 (en) * | 2002-03-01 | 2005-05-05 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-Ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments |
US8568799B2 (en) | 2002-03-22 | 2013-10-29 | Unigen, Inc. | Isolation of a dual COX-2 and 5-lipoxygenase inhibitor from acacia |
US9168242B2 (en) | 2002-03-22 | 2015-10-27 | Unigen, Inc. | Isolation of a dual COX-2 and 5-lipdxygenase inhibitor from Acacia |
US20030232763A1 (en) * | 2002-04-30 | 2003-12-18 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent |
US8945518B2 (en) | 2002-04-30 | 2015-02-03 | Unigen, Inc. | Formulation of dual eicosanoid system and cytokine system inhibitors for use in the prevention and treatment of oral diseases and conditions |
US20060079467A1 (en) * | 2002-04-30 | 2006-04-13 | Unigen Pharmaceuticals, Inc. | Formulation of dual eicosanoid system and cytokine system inhibitors for use in the prevention and treatment of oral diseases and conditions |
US9655940B2 (en) | 2002-04-30 | 2017-05-23 | Unigen, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent |
US20060177528A1 (en) * | 2002-04-30 | 2006-08-10 | Unigen Pharmaceuticals, Inc. | Formulation Of A Mixture Of Free-B-Ring Flavonoids And Flavans As A Therapeutic Agent |
US9849152B2 (en) | 2002-04-30 | 2017-12-26 | Unigen, Inc. | Formulation of a mixture of Free-B-ring flavonoids and flavans as a therapeutic agent |
US7695743B2 (en) | 2002-04-30 | 2010-04-13 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-Ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments |
US20080096827A1 (en) * | 2002-04-30 | 2008-04-24 | Unigen Pharmaceuticals, Inc. | Formulation Of A Mixture Of Free-B-Ring Flavonoids And Flavans For Use In The Prevention And Treatment Of Cognitive Decline And Age-Related Memory Impairments |
US20080096826A1 (en) * | 2002-04-30 | 2008-04-24 | Unigen Pharmaceuticals, Inc. | Formulation Of A Mixture Of Free-B-Ring Flavonoids And Flavans For Use In The Prevention And Treatment Of Cognitive Decline And Age-Related Memory Impairments |
US9370544B2 (en) | 2002-04-30 | 2016-06-21 | Unigen, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent |
US8652535B2 (en) | 2002-04-30 | 2014-02-18 | Unigen, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments |
US20030216481A1 (en) * | 2002-04-30 | 2003-11-20 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-ring flavonoids and flavans as a therapeutic agent |
US7674830B2 (en) | 2002-04-30 | 2010-03-09 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent |
US7514469B2 (en) | 2002-04-30 | 2009-04-07 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of Free-B-ring flavonoids and flavans as a therapeutic agent |
EP2108370A1 (en) | 2002-04-30 | 2009-10-14 | Unigen Pharmaceuticals, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent |
US8034387B2 (en) | 2002-04-30 | 2011-10-11 | Unigen, Inc. | Formulation of a mixture of free-B-ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments |
US6599539B1 (en) * | 2002-07-31 | 2003-07-29 | Poulenger Usa Inc. | Perilla seed pesticide |
US20060078630A1 (en) * | 2003-02-26 | 2006-04-13 | Schempp Christoph M | Method for the production of flavonoid-containing compositions and use thereof |
US7972632B2 (en) | 2003-02-28 | 2011-07-05 | Unigen Pharmaceuticals, Inc. | Identification of Free-B-Ring flavonoids as potent COX-2 inhibitors |
US20070135359A1 (en) * | 2003-02-28 | 2007-06-14 | Unigen Pharmaceuticals, Inc. | Identification of Free-B-Ring Flavonoids as Potent COX-2 Inhibitors |
US20040191327A1 (en) * | 2003-03-24 | 2004-09-30 | Council Of Scientific And Industrial Research | Method of treating and/or preventing asthma using natural compound luteolin |
US20110207806A1 (en) * | 2003-04-04 | 2011-08-25 | Unigen, Inc. | Formulation of dual cycloxygenase (cox) and lipoxygenase (lox) inhibitors for mammal skin care |
US20040220119A1 (en) * | 2003-04-04 | 2004-11-04 | Unigen Pharmaceuticals, Inc. | Formulation of dual cycloxygenase (COX) and lipoxygenase (LOX) inhibitors for mammal skin care |
US9622964B2 (en) | 2003-04-04 | 2017-04-18 | Unigen, Inc. | Formulation of dual cycloxygenase (COX) and lipoxygenase (LOX) inhibitors for mammal skin care |
CN1798568B (en) * | 2003-04-04 | 2011-05-11 | 尤尼根制药公司 | Formulation of dual cycloxygenase (cox) and lipoxygenase (lox) inhibitors for mammal skin care |
US8790724B2 (en) | 2003-04-04 | 2014-07-29 | Unigen, Inc. | Formulation of dual cycloxygenase (COX) and lipoxygenase (LOX) inhibitors for mammal skin care |
US7368249B2 (en) | 2003-07-24 | 2008-05-06 | The Curators Of The University Of Missouri | Methods and compositions for evaluation and modulation of fertility |
US20050112699A1 (en) * | 2003-07-24 | 2005-05-26 | Peter Sutovsky | Methods and compositions for evaluation and modulation of fertility |
US7384654B2 (en) | 2004-02-05 | 2008-06-10 | Access Business Group International Llc | Anti-Allergy composition and related method |
US20060134236A1 (en) * | 2004-02-05 | 2006-06-22 | Access Business Group International Llc | Anti-allergy composition and related method |
US7384656B2 (en) | 2004-02-05 | 2008-06-10 | Access Business Group International Llc | Anti-allergy composition and related method |
CN100438867C (en) * | 2004-03-05 | 2008-12-03 | 惠汝太 | Application of flavone monomer in antivirus drug preparing process |
CN100569241C (en) * | 2007-01-17 | 2009-12-16 | 北京天川军威医药技术开发有限公司 | Celery seed acetic acid ethyl ester extract and uses thereof |
EP4008327A1 (en) | 2009-04-29 | 2022-06-08 | Amarin Pharmaceuticals Ireland Limited | Pharmaceutical compositions comprising epa and a cardiovascular agent and methods of using the same |
EP3791880A1 (en) | 2009-04-29 | 2021-03-17 | Amarin Pharmaceuticals Ireland Limited | Pharmaceutical compositions comprising epa |
US9670165B2 (en) | 2012-03-13 | 2017-06-06 | Dana-Farber Cancer Institute, Inc. | SIK inhibitor for use in a method of treating an inflammatory and/or immune disorder |
US10233157B2 (en) | 2012-03-13 | 2019-03-19 | Dana-Farber Cancer Institute, Inc. | SIK inhibitor for use in a method of treating an inflammatory and/or immune disorder |
WO2013136070A1 (en) * | 2012-03-13 | 2013-09-19 | University Court Of The University Of Dundee | A sik inhibitor for use in a method of treating an inflammatory and/or immune disorder |
CN103099845A (en) * | 2013-02-25 | 2013-05-15 | 中国农业科学院农业质量标准与检测技术研究所 | Method for extracting perillaseed hydrophilic chemical component from perillaseed oil residue |
US10752581B2 (en) | 2013-10-10 | 2020-08-25 | Eastern Virginia Medical School | 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase |
US10266488B2 (en) | 2013-10-10 | 2019-04-23 | Eastern Virginia Medical School | 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase |
US11274077B2 (en) | 2013-10-10 | 2022-03-15 | Eastern Virginia Medical School | 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase |
US10457691B2 (en) | 2014-07-21 | 2019-10-29 | Dana-Farber Cancer Institute, Inc. | Macrocyclic kinase inhibitors and uses thereof |
US10287268B2 (en) | 2014-07-21 | 2019-05-14 | Dana-Farber Cancer Institute, Inc. | Imidazolyl kinase inhibitors and uses thereof |
US10975058B2 (en) | 2014-07-21 | 2021-04-13 | Dana-Farber Cancer Institute, Inc. | Imidazolyl kinase inhibitors and uses thereof |
US10265321B2 (en) | 2014-08-08 | 2019-04-23 | Dana-Farber Cancer Institute, Inc. | Uses of salt-inducible kinase (SIK) inhibitors |
CN106333994B (en) * | 2015-07-10 | 2020-04-17 | 财团法人医药工业技术发展中心 | Perilla seed extract and pharmacological action thereof |
CN106333994A (en) * | 2015-07-10 | 2017-01-18 | 财团法人医药工业技术发展中心 | Perilla seed extract and pharmacological effect thereof |
US10954242B2 (en) | 2016-07-05 | 2021-03-23 | The Broad Institute, Inc. | Bicyclic urea kinase inhibitors and uses thereof |
US11725011B2 (en) | 2016-07-05 | 2023-08-15 | The General Hospital Corporation | Bicyclic urea kinase inhibitors and uses thereof |
US11241435B2 (en) | 2016-09-16 | 2022-02-08 | The General Hospital Corporation | Uses of salt-inducible kinase (SIK) inhibitors for treating osteoporosis |
US11285158B2 (en) | 2017-02-28 | 2022-03-29 | The General Hospital Corporation | Uses of pyrimidopyrimidinones as SIK inhibitors |
US11878019B2 (en) | 2017-02-28 | 2024-01-23 | The General Hospital Corporation | Uses of pyrimidopyrimidinones as SIK inhibitors |
Also Published As
Publication number | Publication date |
---|---|
JP4231559B2 (en) | 2009-03-04 |
JPH10298098A (en) | 1998-11-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6217875B1 (en) | Inhibitors of lipoxygenase | |
KR102126470B1 (en) | Cosmetic Composition for comprising longanae arillus extracts | |
JP2000086510A (en) | Histamine release inhibitor | |
KR20130060611A (en) | Anti-inflammatory composition using a flower extract of citrus unshiu | |
KR101230277B1 (en) | Composition of External Application for Using Bamboo Sprout | |
JP2002121145A (en) | Xanthine oxidase inhibitor | |
US7511078B2 (en) | Antiallergic agents, drugs, foods, drinks or cosmetics containing them and process for producing the same | |
WO2011115061A1 (en) | Hyaluronic acid production promoter | |
JP4420358B1 (en) | Hyaluronic acid production promoter | |
CN110072516B (en) | Cosmetic composition comprising extracts of Chinese medicinal materials as effective ingredients | |
KR20180041887A (en) | Composition for preventing, improving or treating atopic dermatitis comprising extract mixture of Diospyros lotus leaf and grape fruit stem as effective component | |
JP2010024222A (en) | Collagen production promoter, hyaluronic acid production promoter, and collagen production and hyaluronic acid production promoter | |
KR101455842B1 (en) | Anti-Inflammation Composition and an Improvment Composition of Atopic Dermatitis Using an Extract of Immature Fruit of Diospyros kaki | |
KR101501538B1 (en) | Composition for External Application to the Skin Using an Extract of Seeds of Opuntia ficus-indica var. saboten | |
KR20180114629A (en) | Composition for anti inflammation comprising extract of ginseng floral axis | |
JP2009024023A (en) | Lipoxygenase inhibitor | |
KR102221627B1 (en) | Composition comprising Rhus Semialata extract as active ingredient | |
JP2003012532A (en) | Hyaluronidase inhibitor, hexosaminidase liberation inhibitor, cyclic amp phosphodiesterase inhibitor and cosmetic for ameliorating skin roughening | |
JP3385293B2 (en) | Altocarpine-containing antibacterial and preservatives and cosmetics | |
JP5596836B2 (en) | MIF secretion inhibitor | |
US7431953B2 (en) | Skin preparation for external use containing Purpuricenus temminckii frass as the active ingredient | |
KR102105672B1 (en) | Cosmetic Composition for comprising longanae arillus extracts | |
KR20180069528A (en) | Cosmetic Composition for comprising longanae arillus extracts | |
JP6723979B2 (en) | Wrinkle improver | |
KR101332531B1 (en) | Composition for preventing and/or treating itching containing component originating in the bark of tree belonging to the genus acacia |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ORYZA OIL & FAT CHEMICAL CO., LTD., JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MURAI, HIROMICHI;OKADA, TADASHI;YAMAMOTO, HIROYO;REEL/FRAME:009087/0392 Effective date: 19980325 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
FPAY | Fee payment |
Year of fee payment: 8 |
|
FPAY | Fee payment |
Year of fee payment: 12 |